Beet molasses fermentation



Patented Feb. 1, 1938 BEET MOLASSES FERIVIENTATIGN of Maryland NoDrawing. Application July so, 1934, Serial No. 737,575

9 Claims.

The present invention relates to the production of valuable products bythe fermentation of beet molasses. More particularly, this inventionrelates to the butyl alcohol fermentation of beet molasses by means ofbacteria of the general type Clostridium saccharo-acetobutylicum.

The fermentation of soluble carbohydrate mashes, in general, by means ofbacteria of the group Clostridium saccharo-acetobutylicum has beendisclosed in copending application U. S. Ser. No. 675,459 by J. C.Woodrufi, D. A. Legg, and H. R. Stiles, filed June 12, 1933. Accordingto the process of this application, soluble carbohydrate mashes, such ascane molasses mashes, containing ammonia nitrogen and preferably, also,degraded protein nitrogen, are fermented while controlling the acidityof the mash by the addition of alkaline neutralizing agents whereby thefinal hydrogen ion concentration falls within the range pH 5.0 to pH6.2. This process gives very satisfactory yields with most solublecarbohydrate mashes, but we have found that in the case of beet molassesmashes the yields are unsatisfactorily low, in most cases sufficientlyso to make the process impractical from a commercial standpoint.

In view of the unusually high nitrogenous content of beet molasses (from4 to 5 times that of cane molasses) it was thought that an excessiveconcentration of nitrogenous materials might be responsible for thedecreased yield. The elimination of the additional ammonia nitrogen ofU. S. Ser. No. 675,459 succeeded in increasing the yield somewhat, butdid not give rise to satisfactory commercial yields.

We have now made the surprising discovery that very satisfactory yieldsmay be obtained if additional nitrogen rather than less nitrogen isemployed, prov ding a specific type of nitrogenous nutrient is used andthere is present in the mash an adequate supply of available phosphate.The type of nitrogenous nutrient which we have found to be suitable isexemplified by the meal obtained from the corn germ after the expressionof oil therefrom. This material is known as corn germ meal or oil cakemeal. The phosphates which are employed in conjunction with thenitrogenous nutrient may be any of the soluble non-toxic phosphatesgenerally employed as mineral nutrients in fermentation mashes. Alkaliphosphates including ammonium'phosphate, soluble complex alkaline earthphosphates, and the free acid which may be termed hydrogen phosphate,are all suitable for this purpose. As specific examples there may bementioned (NI-I4)2HPO4 NaZHPO i, K2HPO4, superphosphate (CaHdPOr)2.1120) and the like.

The amount of corn germ meal to be employed in any case will of coursedepend upon the concentration and state of degradation of the otherother soluble carbohydrate mashes.

nitrogenous material present in the mash. For example, a mash containingsubstantial amounts of cane molasses, or other materials containingsubstantial concentrations of complex nitrogenous matter, will requireless additional nitrogenous nutrient than one in which beet molassescomprises substantially all of the fermentable carbohydrate. However, ingeneral it may be said that from 1 to 4% by weight of corn germ meal,based on the weight of molasses used, will usuallybe found to besatisfactory. From '2-3% will generally be found to be preferable andmay advantageously be employed in any mash containing beet molasses as amajor source of carbohydrate. In any case, the lower economical limit ofgerm meal concentration may easily be determined by preliminaryfermentations.

The concentration of phosphate to be employed will likewise depend uponthe available phosphate in the mash. Most samples of beet molasses willbe found to be deficient in suitable phosphate, but in occasionalsamples optimum yields may be obtained without the addition of furthermineral nutrients. The amount of supplementary phosphate to be addedwill also depend upon the amount introduced in other materials in themash. For example, a mixed cane and beet molasses mash will require lessadditional mineral nutrient than one in which beet molasses comprisessubstantially the whole source of carbohydrate. In general, however, itmay be said that from 0.01% to 0.15% by weight of a soluble phosphatewill be satisfactory. From 0.03% to 0.10% will generally be. found to bepreferable and may be satisfactorily employed in any mash containingbeet molasses as a major component. The exact concentration to be usedfor optimum results may, of course, readily be determined by preliminaryfermentations.

An important aspect of the fermentation of beet molasses mashescomprises the acidity control during the fermentation. We have foundthat the organisms of the type Clostrz'dium saccharo-acetobutylicumgenerally require a more alkaline reaction for the'fermentation of beetmolasses mashes than for the fermentation of In copending application U.S. Ser. No. 675,459 it is stated that for the usual type of solublecarbohydrate mash the acidity should be controlled so that the finalhydrogen ion concentration falls within the range pH 5.0 to pH 6.2.However, we have found that for mashes containing beet molasses as amajor component the acidity of the fermenting mash should be maintainedat a value such that the final hydrogen ion concentration falls withintherange pH 5.5 to 7.0 and preferably within the range pH 5.7 to 6.5.

The usual types of beet molasses will be found to contain considerablequantities of alkaline buffering materials and to have an initialalkaline reaction ranging from pH 7.0 to pH 9.0. This degree ofalkalinity is usually insufiicient to adversely effect the fermentation,and the mashes may be inoculated at their original hydrogen ionconcentration without the necessity for neutralizing. In fact, we havefound that it is generally undesirable to attempt to neutralize themashes with strong acids such as mineral acids. However, if thephosphate is supplied in the form of a free acid, this amount of acid isinsuificient to adversely effect the fermentation; and considerableamounts of organic acids may also be introduced without undesirableresults.

The alkaline buffering capacity of various samples of beet molasses willbe found to differ to some extent, but in many cases it will be found tobe sufiicient to maintain the acidity within the necessary limits tosecure the desired final hydrogen ion concentration. In the case ofsamples of molasses which are deficient in buffering materials, or incase unduly acidic materials are introduced into the mash, the acidityof the fermenting mash may be controlled by any of the methods disclosedin copending application Ser. No. 675,459. For example, the fermentationmay be continuously or semi-continuously neutralized with solublealkalies, or an insoluble alkaline material may be incorporated into themash before inoculation. In any case, the necessity for the use ofalkaline neutralizing materials and the amounts necessary for optimumyields may readily be determined by preliminary fermentations.

The organisms which are suitable for use in our process are the bacteriaof the group Clostridium saccharo-acetobutylicum, which are described atlength in copending application U. S. Ser. No. 675,459, referred toabove. Our process is particularly adapted to fermentations by means ofClostridz'um saccharo-acetobutylicum on which is likewise described indetail in said copending application. Other members of this group ofbacteria, such as C'lostridium saccharo-acetobutylzcum 3 andClostrz'clium saccharo-acetobutylicum 'y described in copendingapplication Ser. No. 714,633, now Patent No. 2,050,219, issued Aug. 4,1936, may likewise suitably be employed.

Our invention may perhaps best be illustrated by the following specificexamples.

Example I Sterile mashes containing approximately 10% 01' Ohio beetmolasses, with and without corn germ meal and phosphates, wereinoculated with an actively fermenting culture of CZostridiumsaccharo-acetobutylicum oz and incubated at 30 C. for 68 hours. Theresults of these fermentations are shown in the table below:

Corn Yield Sugar germ g g concenmeal Type of concen trationcoucenphosphate nation Percent Grams percent tration ercent of totalsolvents percent p sugar per liter 4.94 0.00 0.00 21. 1 10.4 5.08 0. 00(NHOzHPOr- 0.03 29.0 14. 7 5. 02 0. 00 NBzHPOl. 0. 10 29. 1 14. 6 4. 980. 25 I 0. 00 36. 5 18. 2 5. 04 0. 25 (NHOzHPO-r- 0. 03 37. 5 18. 9 5.04 0. 25 NazHPO4 0. 10 37. 9 19. 1

Example II Sterile mashes containing approximately 10% of Michigan beetmolasses, with and without corn germ meal and diammonium phosphate, wereinoculated with an actively fermenting culture of Clostridiumsaccharo-acetobutylicum oz and incubated at 30 C. for 68 hours. Theresults of these fermentations are shown in the table below:

Yield Corn germ Essa; m1

ercent ccntration Percent Grams p percent D of total solvents sugar perliter Example III A sterile mash containing 4.86% of sugar as Michiganbeet molasses (from a different locality from that of Example II), 0.25%by weight of corn germ meal, and no additional phosphate was inoculatedwith an actively fermenting culture of Clostridiumsaccharo-acetobutylicum a and incubated at 30 C. for. 68' hours. Theyield and solvent ratio were found to be as follows:

Yield Solvent ratio Grams solvents per liter Percent of Butyl totalalcohol Ethyl Acetone alcohol sugar It may be seen from the aboveresults that a full yield was obtained in this case without the additionof supplementary phosphate, indicating that this particular sample ofmolasses contained sufficient phosphate for this type of fermentation.

Example IV Sterile mashes containing approximately 10% of Wisconsin beetmolasses, with and without corn germ meal and diammonium phosphate, wereinoculated with an actively fermenting culture of Clostridiumsaccharo-acetobutylicum 0c and incubated at 30 C. for 68 hours. Theresults of these fermentations are shown in the table below:

- Yield Corn germ Sugar con- Concentration centration mealcancer" of (NHOQHPO;

tration per- Percent Grams percent cent percent of total solvents sugarper liter It may be seen from the above table that this particularsample of molasses was apparently strongly deficient in phosphate, butrequired little supplementary nitrogenous nutrient. It will of course beevident to those skilled in the art that samples from various sourceswill differ in their nutrient deficiencies and that optimum results canbe most economically secured only after preliminary fermentations todetermine the requirements for the particular sample.

Although our invention is illustrated by the above specific examples, itis to be distinctly understood that it is not limited to the particularmaterials or procedures described therein.

arc-mace For example, mixed meshes containing cane molasses, hydroi, orother soluble carbohydrate, in addition to the beet molasses, may besuccess fully employed in this fermentation. Our process is applicable,generally, to soluble carbohydrate mashes containing beet molasses as amajor component, irrespective of the remaining carbohydrate content.Likewise, it will be apparent to those skilled in the art that variousnitrogenous materials are equivalent to corn germ meal in proteinaceouscontent and state of degradation and may be used with equal success inthe present process. Various modifications of procedure may also beemployed without departing from the scope of our invention. For example,if a mixed mash is to be employed, such as a mixed cane and beetmolasses mash, the fermentation could suitably be started in the canemolasses and the beet molasses and supplementary nutrients added to thismash after fermentation had become sumciently active. the variousmodifications of procedure disclosed in copending application Ser. No.675,459 will likewise be apparent to one skilled in the art. In general,it may be said that any such modifications or the use of any equivalentswhich would naturally occur to a skilled bacteriologist or fermentationchemist may be employed without departing from the'scope of ourinvention.

Our invention now having been described, what we claim is:

I. In the fermentation of an essentially soluble carbohydrate mashcontaining beet molasses as a major component, by means of bacteria ofthe group Clostridium saccharo-acetobutylicum, the step which compriseseffecting the fermentation in a mash containing com germ meal and asoluble non-toxic phosphate.

2. In the fermentation of an essentially soluble carbohydrate mashcontaining beet molasses as a major component, by means of bacteria ofthe group Clostridium saccharo-acetobutylicum, the step which comprisesefiecting the fermentation in a mash containing from 0.1% to 0.4% ofcorn germ meal and from 0.01% to 0.15% of a soluble non-toxic phosphate.

3. In the fermentation of an essentially soluble carbohydrate mashcontaining beet molasses as a major component, by means of bacteria ofthe group Clostridium saccharo-acetobutylicum, the step which compriseseffecting the fermentation in a mash containing from 0.2% to 0.3% ofcorn germ meal and from 0.03% to 0.10% of a soluble non-toxic phosphate.

4. In the fermentation of an essentially soluble carbohydrate mashcontaining beet molasses as a major component, by means of bacteria ofthe group Clostridium sacchcro-acetobutylicum, the step which compriseseffecting the fermentation ina mash containing corn germ meal and amin-.-

The applicability of ble non-toxic phosphate, and maintaining theacidity of the fermenting mash at a value such that the final hydrogenion concentration falls within the range pH 5.5 to 7.0.

5. In the fermentation of an essentially soluble carbohydrate mashcontaining beet molasses as a major component, by means of bacteriaofthe group Clostridium saccharo-acetobutylicum, the step which comprisesefiecting the fermentation in a mesh containing from 0.1% to 0.41% ofcorn germ meal and from 0.01% to 0.15% .of a soluble non-toxicphosphate, and maintaining the acidity of the fermenting mash at a valuesuch that the final hydrogen ion concentration falls within the range pH5.5 to 7.0.

6. In the fermentation of an essentially soluble carbohydrate mashcontaining beet molasses as a major component, by means of bacteria ofthe group Clostridium saccharo-acetobutylicum, the step which compriseseffecting the fermentation in a mash containing from 0.2% to 0.3% ofcorn germ meal and from 0.03% to 0.10% of a soluble non-toxic phosphate,and maintaining the acidity of the fermenting mash at a value such thatthe final hydrogen ion concentration falls within the range pH 5.5 to7.0.

'7. In the fermentation of an essentially soluble carbohydrate mashcontaining beet molasses as a major component, by means of bacteria ofthe group Clostridium saccharo-acetobutylicum, the step which comprisesefiecting the fermentation in a mash containing corn germ meal and asoluble non-toxic phosphate, and maintainingthe acidity of thefermenting mash at a value such that the final hydrogen ionconcentration falls within the range pH 5.7 to 5.5.

8. In the fermentation of an essentially soluble carbohydrate mashcontaining beet molasses as a major component, by means of bacteria ofthe group Clostridium saccharmacetobutylicum, the step which comprisesefiecting the fermentation in a mash containing from 0.1% to 0.4% ofcorn germ meal and from 0.01% to 0.15% of a soluble non-toxic phosphate,and maintaining the acidity of the fermenting mash at a value such thatthe final hydrogen ion concentration falls within the range pH 5.? to6.5. g

9. In the fermentation of an essentially soluble carbohydrate mashcontaining beet molasses as a major component, by means of bacteria ofthe group Clostridium saccharo-acetobutylicum, the step which compriseseflecting the fermentation in a mash containing from 0.2% to 0.3% ofcorn germ meal and from 0.03% to 0.10% of a soluble non-toxic phosphate,and maintaining the acidity of the fermenting mash at a value such thatthe final hydrogen ion concentration falls within the range pH 5.7 to6.5.

DAVID A. LEGG. NOBLE R. TARVIN.

